The tumor suppressor p53 is mutated in over 50% of all human cancers. Increase of p53 activity in mice leads to remarkable resistance to cancer formation, but no extension of life span. Surprisingly, expression of hyperactive forms of p53 in mice actually leads to shortened life spans. Mice with hyperactive p53 show several signs of accelerated aging, which leads to the hypothesis that reduction of p53 activity could extend life span if tumor formation can be avoided. To investigate this hypothesis we use the fruit fly Drosophila melanogaster as it consists of mostly post-mitotic tissue and is thus largely tumor resistant. In fact, flies that express dominant-negative versions of p53 in neurons live up to 58% longer. This proposal aims to elucidate the context of the biochemical signaling pathways that p53 is involved in to regulate longevity. My data shows that the life span extending effect of dominant-negative p53 is not additive to the life span extending effects of calorie restriction or dSir2 over expression. Furthermore, over expressed fly p53 physically interacts with dSir2 and peptides derived from fly p53 are efficiently deacetylated by dSir2. This suggests that p53 may mediate aspects of the calorie restriction life span extension pathway down stream of dSir2. However, the events following p53 inactivation still remain to be elucidated. Preliminary data show that expression of dominant-negative p53 in a set of only fourteen insulin-producing neurons is sufficient to extend life span. In addition, these flies have reduced levels of insulin-like peptide 2 and show a reduction in insulin-like signaling activity. Interestingly, the fourteen insulin-producing neurons are the functional equivalent of mammalian pancreatic p-cells. These data suggest a connection between p53 and insulin signaling. The experiments proposed here will therefore test the hypothesis that life span extension through dominant-negative p53 is related to the insulin/insulin-like growth factor pathway of life span extension. Aim 1 will measure a variety of determinants of energy homeostasis and compare the metabolic state of p53 long-lived flies to that of insulin-signaling long-lived flies. Aim 2 will establish whether specific molecular changes that occur in insulin-signaling long-lived flies also happen in p53 long-lived flies. These changes include levels in insulin mRNA, PI3K activity status and dFoxO activity status. Aim 3 will investigate genetically if the p53 pathway and the insulin-signaling pathway interact or are two different longevity pathways. Relevance: The tumor suppressor p53 has been shown to also be a major regulator of longevity. This proposal aims to uncover the signaling mechanisms that lead to p53-dependent life span regulation and will thus shed light on the mechanisms of calorie restriction as well.